Novel solid forms of tacedinaline

ABSTRACT

Novel solid forms of tacedinaline (4-(acetylamino)-N-(2-aminophenyl)benzamide), including crystalline tacedinaline Form B, a novel crystalline tacedinaline TFA salt, and amorphous tacedinaline, are disclosed. Pharmaceutical compositions comprising crystalline tacedinaline Form B, the novel crystalline tacedinaline TFA salt, and/or amorphous tacedinaline, and methods of treating various conditions by administering those novel solid forms, are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority under 35 U.S.C. §119 to U.S.Provisional Application 61/360,271, filed Jun. 30, 2010, which isincorporated herein by reference.

TECHNICAL FIELD

The invention relates to novel solid forms of tacedinaline,pharmaceutical compositions comprising the novel solid forms, andmethods of treating and/or preventing various conditions byadministering the novel solid forms.

BACKGROUND

The solid form (i.e., the crystalline or amorphous form) of apharmaceutical compound can be important relative to its pharmacologicalproperties and development as a viable active pharmaceutical ingredient(“API”).

Pharmaceutical products are often formulated from crystalline compoundsbecause crystalline materials may provide higher levels of purity andresistance to physical and chemical instabilities under ambientconditions, relative to amorphous forms. Crystalline forms of a compoundmay in some cases, offer advantages over amorphous forms, such asimproved solubility, stability, processing improvements, etc., anddifferent crystalline forms (e.g. polymorphs of the compound) may offergreater or lesser advantages over one another. However, crystallineforms of a compound are not predictable, and in fact, are not alwayspossible. It is a well-accepted principle that the formation of a newpolymorphic or crystalline form (e.g. a new crystalline salt form) of acompound is totally unpredictable, and until a particular polymorph isprepared, there is no way to know whether it might exist, how to prepareit, or what its properties might be. Bernstein, J. Polymorphism inMolecular Crystals. New York: Oxford University Press, 0.9 (2002).

Unlike a crystalline solid, which has an orderly array of unit cells inthree dimensions, amorphous forms lack long-range order becausemolecular packing is more random. As a result, amorphous organiccompounds tend to have different properties than their crystallinecounterparts. For example, amorphous compounds often have greatersolubility than crystalline forms of the same compound. Thus, by way ofexample only, in pharmaceutical formulations whose crystalline forms arepoorly soluble, amorphous forms may present attractive formulationoptions. As such, amorphous APIs may be used to improve physical andchemical properties of drugs, such as, for example, dissolution andbioavailability.

Solid forms of a compound, including both crystalline and amorphousforms, are of particular interest to the pharmaceutical industry, forexample to those involved in the development of suitable dosage forms,if the solid form of the API (e.g. the crystalline polymorphic form oramorphous form) is not held constant during clinical or stabilitystudies, the exact dosage form used or studied may not be comparablefrom one lot to another. In addition, regulatory agencies require solidform characterization and control of the API for approval. Certainpolymorphic forms may exhibit enhanced thermodynamic stability or may bemore readily manufactured in high purity in large quantities, and thusare more suitable for inclusion in pharmaceutical formulations. Certainpolymorphs may display other advantageous physical properties such aslack of hygroscopic tendencies, improved solubility, and enhanced ratesof dissolution due to different lattice energies. As such, finding theright conditions to obtain a particular solid form of the desired API(e.g. a particular crystalline polymorphic form or an amorphous form),with pharmaceutically acceptable properties, is critical to drugdevelopment, but can take significant time, resources, and effort.

Tacedinaline, 4-(acetylamino)-N-(2-aminophenyl)benzamide, (shown below)is a known API useful for treating and/or preventing a variety ofconditions, such as, for example, combating neoplastic diseases, and isrecognized as an HDAC inhibitor.

For example, tacedinaline has positive indications for the treatment ofprostate cancer. The preparation and pharmacologic activity oftacedinaline are described in, for example, U.S. Pat. No. 5,137,918, WO2009/076234, Gediya, L. K. et al., Bioorganic & Medicinal Chemistry2008, 16, 3352-3360; and Thomas, M. et al., Bioorganic & Medicinalchemistry 2008, 16, 8109-8116, all of which are incorporated herein byreference.

While therapeutic efficacy is a primary concern for a therapeutic agentsuch as tacedinaline, as discussed above the solid form of apharmaceutical drug candidate is also important. For example, each solidform of a drug candidate can have different solid state (physical andchemical) properties. The differences in physical properties exhibitedby a different solid form of an API, such as a polymorph of the originalcompound, can affect pharmaceutical parameters such as storagestability, compressibility and density, all of which may be important informulation and product manufacturing, and solubility and dissolutionrates, which may be important factors in determining bioavailability.Because these practical physical properties can be influenced by thesolid form of the API, they can significantly impact the selection of acompound as an API, the ultimate pharmaceutical dosage form, theoptimization of manufacturing processes, and absorption in the body.Moreover, finding the most adequate form for further drug developmentcan reduce the time and the cost of that development. It may also bebeneficial to identify and characterize additional crystal forms so thatthey may be recognized if they appear during drug development and/ormanufacturing.

Obtaining pure solid forms, then, can be extremely useful in drugdevelopment, as it generally permits better characterization of the drugcandidate's chemical and physical properties. Crystalline forms oftenhave more favorable chemical and physical properties than amorphousforms of the same compound. As such, one or more crystalline forms maypossess more favorable pharmacology than amorphous forms or be easier toprocess, or may have better storage stability. Similarly, onecrystalline form may possess more favorable pharmacology, may be easierto process, or may have better storage stability than another, or thanan amorphous form, or vice versa.

One such physical property is a pharmaceutical compound's dissolutionrate in aqueous fluid. The rate of dissolution of an API in a patient'sstomach fluid may have therapeutic consequences since it impacts therate at which an orally administered active ingredient may reach thepatient's bloodstream.

Another such physical property is thermodynamic stability. Thethermodynamic stability of an active ingredient may have consequences onthe manufacturing process and storage stability of the API and/or theformulation.

A crystalline form of a compound generally possesses distinctcrystallographic and spectroscopic properties when compared to othercrystalline forms having the same chemical composition. Crystallographicand spectroscopic properties of the particular form are typicallymeasured by one or more techniques such as x-ray powder diffraction(XRPD), single crystal x-ray crystallography, solid state NMRspectroscopy, infrared spectroscopy (IR), or Raman spectroscopy, amongother techniques. A particular solid form of a compound may oftenexhibit distinct thermal behavior as well. Thermal behavior is measuredin the laboratory by such techniques as capillary melting point,thermogravimetric analysis (TGA), and differential scanning calorimetry(DSC).

Referenced above, U.S. Pat. No. 5,137,918 describes the synthesis andbasic activities of a family of compounds including tacedinaline. Thetacedinaline disclosed therein is reported as having a melting point of243.7° C.

Accordingly, there is a need in the art to identify novel solid forms oftacedinaline, particularly those having advantageous chemical and/orphysical properties. This invention answers those needs by providingnovel solid forms of tacedinaline, including forms having improvedproperties.

SUMMARY

In accordance with various embodiments of the invention and afterextensive experimentation are disclosed novel crystalline forms oftacedinaline, including the three forms referred to herein as Forms A,B, and D, and a novel crystalline tacedinaline TFA salt form.

The invention in various embodiments also relates to pharmaceuticalcompositions and formulations comprising the novel crystalline forms,and methods of treating and/or preventing various conditions byadministering the novel crystalline forms.

In further embodiments, the invention relates to a novel amorphous formof tacedinaline, as well as pharmaceutical compositions and formulationscomprising the novel amorphous form, and methods of treating and/orpreventing various conditions by administering the novel amorphous form.

As used herein, the term “polymorph” refers to different crystallineforms of the same compound and other solid state molecular forms,including pseudopolymorphs. The terms “pseudopolymorph” and“pseudomorph” as used herein are interchangeable and are meant toinclude hydrates (i.e., water present in the crystalline structure) andsolvates (i.e., solvents other than water) of the compound, of both afixed or stoichiometric and variable nature. Different crystallineforms, such as polymorphs, have different crystal structures due to adifferent packing of the molecules in the lattice. This results in adifferent crystal symmetry and/or unit cell parameters which directlyinfluences the physical properties of the form, including X-raycharacteristics (both single-crystal and XRPD) of crystals or powders. Adifferent polymorph, for example, will in general diffract at adifferent set of angles and will give different values for theintensities. Therefore, when available, X-ray techniques can be used toidentify different polymorphs, or a solid form that comprises more thanone polymorph, generally in a reproducible and reliable way, S. Byrn etal., “Pharmaceutical Solids: A Strategic Approach to RegulatoryConsiderations,” Pharmaceutical Research, Vol. 12, No. 7, p. 945-954,1995; J. K. Haleblian and W. McCrone, “Pharmaceutical Applications ofPolymorphism,” Journal of Pharmaceutical Sciences, Vol. 58, No. 8, p.911-929, 1969.

As used herein, the term “XRPD” refers to x-ray powder diffraction.Unless otherwise noted, XRPD analyses were performed either on a ScintagX₁ Advanced Diffraction system or a Rigaku Smart Lab X-ray diffractionsystem.

The Scintag X₁ Advanced Diffraction system is equipped with a VortexSilicon Multi-Cathode detector. Data were collected using Cu Kαradiation. The X-ray tube voltage and amperage were set to 45 kV and 40mA, respectively. The slits used were a 1 mm divergence slit, a 2 mmtube scatter slit, a 0.5 mm detector scatter slit, and a 0.3 mmreference slit. Data were collected in continuous mode from 2 to 40°2θusing a 0.04 degree step and a 2 second collection time per step. Eachspecimen was prepared for analysis by placing it in the 1-mm deep, roundwell of a stainless steel holder and leveling the surface with a glassslide.

The Rigaku Smart-Lab X-ray diffraction system was configured forreflection Bragg-Brentano geometry using a line source X-ray beam. Thex-ray source is a Cu Long Fine Focus tube was operated at 40 kV and 44ma. That source provides an incident beam profile at the specimen thatchanges from a narrow line at high angles to a broad rectangle at lowangles. Beam conditioning slits are used on the line X-ray source toensure that the maximum beam size is less than 10 mm both along the lineand normal to the line. The Bragg-Brentano geometry is a para-focusinggeometry controlled by passive divergence and receiving slits with thespecimen itself acting as the focusing component in the optics. Theinherent resolution of Bragg-Brentano geometry is governed in part bythe diffractometer radius and the width of the receiving slit used.Typically, the Rigaku Smart-Lab is operated to give peak widths of 0.1°2θ or less. The axial divergence of the X-ray beam is controlled by 5.0°Soller slits in both the incident and diffracted beam paths. Each powderspecimen was prepared in a low background Si holder using light manualpressure to keep the sample surface flat and level with the referencesurface of the sample holder. The single crystal Si low backgroundholders have a small circular recess (7 mm diameter and about 1 mindepth) that holds between 5 and 10 mg of powdered material. The standardmeasurement range was from 2 to 40° 2θ using a continuous scan of 3 °2θper minute with an effective step size of 0.02 °2θ.

As used herein, “IR” refers to infrared spectroscopy. Unless otherwisenoted, IR spectra were obtained on a Nicolet 6700 FT-IR system. Sampleswere analyzed using a Nicolet SMART iTR, attenuated total reflectancedevice.

As used herein, “mp” refers to melting point. Melting points weredetermined on a Stuart SMP3 apparatus that was calibrated using acaffeine USP melting point standard. A ramp rate of 1° C./minute wasused.

As used herein, the term “DSC” refers to differential scanningcalorimetry. Unless otherwise noted, DSC data disclosed herein wereobtained using a TA Instruments 2920 instrument. Samples were preparedin crimped aluminum pans and kept under a flow of nitrogen duringanalysis. The heating rate was 10° C./minute.

As used herein, the term “TGA” refers to thermogravimetric analysis.Unless otherwise noted, TGA data disclosed herein were obtained using TAInstruments 2050 instrument. Samples were kept under a flow of nitrogenduring analysis. The heating rate was 10° C./minute.

As used herein, the term “¹H-NMR” refers to proton nuclear magneticresonance spectroscopy. Solution ¹H NMR data disclosed herein wereacquired on a Bruker 300 (300 MHz ¹H) spectrometer. Proton chemicalshifts are reported in ppm, referenced to the NMR solvent. Unlessotherwise indicated, NMR data were collected at 25° C.

As used herein, “LC/MS” refers to tandem liquid chromatography/massspectrometry. LC/MS data referenced herein were acquired on a Waters2795 Alliance HPLC coupled with a Waters 2996 Photodiode Array Detectorand a Waters ZQ Mass Spectrometer. The column was an XBridge 4.6×30 mm,3.5 μm, using a 5 to 95% acetonitrile/water gradient containing 0.01%formic acid over 2.5 minutes. As used herein, “UPLC/MS” refers to tandemUltra Performance Liquid Chromatography/Mass Spectrometer. UPLC/MS datareferenced herein was acquired on a Waters Acquity UPLC coupled with aWaters Acquity PDA Detector and a Waters SQ Mass Spectrometer (singlequadrupole). The column was an Acquity BEH C18 1.0×50 mm, 1.7 um, using5 to 95% acetonitrile/water gradient containing 0.05% trifluoroaceticacid (water) and 0.1% triflouroacetic acid (acetonitrile) over 15minutes.

Single crystal x-ray data reported herein were acquired at lowtemperature (150 K) on a Rigaku Rapid II equipped with confocal optics.Crystals were mounted on a fiber in a random orientation. Additionaldetails are provided below.

As used herein with respect to the various analytical techniquesdescribed herein and data generated therefrom, the terms “substantiallythe same as” or “substantially similar to” is meant to convey that aparticular set of analytical data is, within acceptable scientificlimits, sufficiently similar to that disclosed herein such that one ofskill in the art would appreciate that the crystal form of the compoundis the same as that of the present invention. One of skill in the artwould appreciate that certain analytical techniques, such as, forexample, XRPD, ¹H-NMR, LC/MS, IR, DSC, TGA, and Raman, will not produceexactly the same results every time due to, for example, instrumentalvariation, sample preparation, scientific error, etc. By way of exampleonly, XRPD results (e.g., peak locations, intensities, and/or presence)may vary slightly from sample to sample, despite the fact that thesamples are, within accepted scientific principles, the same crystallineform, and this may be due to, for example, preferred orientation orvarying solvent or water content. It is well within the ability of thoseskilled in the art, looking at the data as a whole, to appreciatewhether such differences indicate the same or a different form, and thusdetermine whether analytical data being compared to those disclosedherein are or are not substantially the same or similar to the solidform it is being compared with.

In this regard, and as is commonly practiced within the scientificcommunity, it is not intended that the exemplary analytical data of thenovel polymorphic forms of tacedinaline disclosed herein be netliterally in order to determine whether comparative data represent thesame form as those disclosed and claimed herein, such as, for example,whether each and every peak of an exemplary XRPD pattern of the novelpolymorphic forms of tacedinaline disclosed herein is present in thecomparative data, in the same location, and/or of the same intensity.Rather, as discussed above, it is intended that those of skill in theart, using accepted scientific principles, will make a determinationbased on the data as a whole regarding whether comparative analyticaldata represent the same or a different form than the novel polymorphicforms of tacedinaline disclosed herein.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show exemplary XRPD patterns of crystalline tacedinalineForm A.

FIG. 2 shows an exemplary IR spectrum of an embodiment of crystallinetacedinaline Form A.

FIG. 3 shows an exemplary TGA profile of an embodiment of crystallinetacedinaline Form A.

FIG. 4 shows an exemplary DSC thermogram of an embodiment of crystallinetacedinaline Form A.

FIGS. 5A-5C show exemplary ¹H-NMR spectra of an embodiment ofcrystalline tacedinaline Form A.

FIG. 6 shows exemplary LC/MS data for an embodiment of crystallinetacedinaline Form A.

FIGS. 7A and 7B show exemplary XRPD patterns of crystalline tacedinalineForm B.

FIG. 8 shows an exemplary IR spectrum of an embodiment of crystallinetacedinaline Form B.

FIG. 9 shows an exemplary TGA profile of an embodiment of crystallinetacedinaline Form B.

FIG. 10 shows an exemplary DSC thermogram of an embodiment ofcrystalline tacedinaline Form B.

FIGS. 11A-11C show exemplary ¹H-NMR spectra of an embodiment ofcrystalline tacedinaline Form B.

FIG. 12 shows exemplary LC/MS data for an embodiment of crystallinetacedinaline Form B.

FIG. 13 shows an exemplary XRPD pattern of an embodiment of a mixture ofcrystalline tacedinaline Forms B and D.

FIG. 14 shows an exemplary IR spectrum of an embodiment of a mixture ofcrystalline tacedinaline Forms B and D.

FIG. 15 shows an exemplary TGA profile of an embodiment of a mixture ofcrystalline tacedinaline Forms B and D,

FIG. 16 shows an exemplary DSC thermogram of an embodiment of a mixtureof crystalline tacedinaline Forms B and D.

FIGS. 17A-17C show exemplary ¹H-NMR spectra of an embodiment of amixture of crystalline tacedinaline Forms B and D.

FIG. 18 shows exemplary LC/MS data for an embodiment of a mixture ofcrystalline tacedinaline Forms B and D.

FIG. 19 is an XRPD pattern of crystalline tacedinaline Form C.

FIG. 20 is an IR spectrum of crystalline tacedinaline Form C.

FIG. 21 is a TGA profile of crystalline tacedinaline Form C.

FIG. 22 is a DSC thermogram of crystalline tacedinaline Form C.

FIGS. 23A-23C are ¹H-NMR spectra of crystalline tacedinaline Form C.

FIG. 24 is LC/MS data for crystalline tacedinaline Form C.

FIG. 25 shows a comparison of exemplary XRPD patterns for crystallinetacedinaline Forms A, B, C, and D, crystalline tacedinaline TFA salt,and amorphous tacedinaline (top to bottom).

FIG. 26 shows an exemplary XRPD pattern of crystalline tacedinaline FormD.

FIG. 27 shows an exemplary IR spectrum of an embodiment of crystallinetacedinaline Form D.

FIG. 28 shows an exemplary TGA profile of an embodiment of crystallinetacedinaline Form D.

FIG. 29 shows an exemplary DSC thermogram of an embodiment ofcrystalline tacedinaline Form D.

FIG. 30 shows an exemplary XRPD pattern of an embodiment of a mixture ofamorphous tacedinaline and N-(4-(1-H-benzo[d]imidazol-2-yl)acetamide.

FIG. 31A shows an exemplary ¹H-NMR spectrum of an embodiment of amixture of amorphous tacedinaline andN-(4-(1-H-benzo[d]imidazol-2-yl)acetamide, and FIG. 31B shows an ¹H-NMRspectrum of N-(4-(1-H-benzo[d]imidazol-2-yl)acetamide.

FIG. 32A shows exemplary LC/MS data of an embodiment of a mixture ofamorphous tacedinaline and N-(4-(1-H-benzo[d]imidazol-2-yl)acetamide,and FIG. 32B shows LC/MS data forN-(4-(1-H-benzo[d]imidazol-2-yl)acetamide.

FIG. 33 shows an exemplary XRPD pattern of an embodiment of crystallinetacedinaline TFA salt.

FIG. 34 shows an exemplary IR spectrum of an embodiment of crystallinetacedinaline TFA salt.

FIG. 35 shows an exemplary TGA profile of an embodiment of crystallinetacedinaline TFA salt.

FIG. 36 shows an exemplary DSC thermogram of an embodiment ofcrystalline tacedinaline TFA salt.

FIG. 37 shows an exemplary ¹H-NMR spectrum of an embodiment ofcrystalline tacedinaline TFA salt.

FIG. 38 shows exemplary LC/MS data for an embodiment of crystallinetacedinaline TFA salt.

FIGS. 39A-39C show an exemplary ¹H-NMR spectrum of an embodiment ofcrystalline tacedinaline Form D.

FIGS. 40A-40C show exemplary UPLC/MS data for an embodiment ofcrystalline tacedinaline Form D.

FIG. 41 shows the Plasma Concentration-Time Curve of crystallinetacedinaline Form A in Sprague-Dawley rats following intravenousinjection and oral administration at 1 mg/kg.

FIG. 42 shows an exemplary DSC thermogram of an embodiment of a mixtureof amorphous tacedinaline and N-(4-(1-H-benzo[d]imidazol-2-yl)acetamide,and FIG. 32B shows LC/MS data forN-(4-(1-H-benzo[d]imidazol-2-yl)acetamide.

FIG. 43 shows a calibration curve of the relative solubilities of FormsA, B, and D using 5 samples of various concentrations in water, as setforth in Table 12 of Example 12.

DETAILED DESCRIPTION

In various embodiments, the invention relates to novel crystalline FormsA, B, and D of tacedinaline, as well as novel amorphous tacedinaline anda novel crystalline tacedinaline TFA salt form. Exemplary methods ofpreparing these novel solid forms are found in the examples below.

In further embodiments, the invention relates to pharmaceuticalcompositions and formulations comprising the novel solid forms oftacedinaline, and methods of treating and/or preventing variousconditions by administering the novel solid forms.

Crystalline Tacedinaline Form A

Crystalline tacedinaline Form A was obtained in a crystalline solid formthat is characterized by a unique XRPD pattern substantially as shown inFIGS. 1A and 1B, and a unique IR spectrum substantially as shown in FIG.2. Crystalline tacedinaline Form A was found to be an anhydrate, assuggested by the representative TGA plot in FIG. 3, exhibiting no weightloss prior to decomposition. The anhydrous nature of tacedinaline Form Awas confirmed by the single crystal structure. Tacedinaline Form A has amelting temperature in the range of about 239-240° C., as found byvisual determination, and exhibits a corresponding endothermic event atabout 247° C., as shown by the representative DSC trace in FIG. 4.Subsequent to this endothermic event, tacedinaline Form A undergoes athermally activated dehydrative intramolecular cyclization to formN-(4-(1-H-benzo[d]imidazol-2-yl)acetamide, which exhibits acharacteristic endothermic event at about 310° C.

An exemplary listing of representative XRPD peaks of an embodiment oftacedinaline Form A can be found in Table 1. An exemplary listing ofrepresentative IR peaks of an embodiment of tacedinaline Form A can befound in Table 2.

Crystalline tacedinaline Form A exhibits improved properties relative toother forms of tacedinaline, including that disclosed in the art (FormC). For example, Form A has improved thermal stability relative totacedinaline Form C, which contains methanol and may be undesirable invarious embodiments. Additionally, Form A is more thermodynamicallystable under certain conditions relative to Forms B and D, as shown inExamples 11 and 12.

TABLE 1 Form A Degrees 2θ 10.28 12.31 13.70 15.69 16.44 17.90 18.3619.04 19.45 19.90 23.55 24.78 25.25 26.95 27.67

TABLE 2 Form A Reciprocal cm 3396.2 3310.3 3247.5 3185.8 3108.0 3048.51690.9 1642.1 1594.2 1528.3 1504.2 1485.6 1456.5 1404.3 1370.4 1308.61283.6 1265.0 1224.6 1177.5 1123.5 1069.5 1037.3 1016.1 1009.5 904.3848.1 796.7 745.9 656.9 601.3

Crystalline Tacedinaline Form B

Crystalline tacedinaline Form B was obtained in a crystalline solid formthat is characterized by a unique XRPD pattern substantially as shown inFIGS. 7A and 7B, and an IR spectrum substantially as shown in FIG. 8.Crystalline tacedinaline Form B was found to be an anhydrate, assuggested by the representative TGA plot in FIG. 9 that exhibits noweight loss prior to decomposition. Tacedinaline Form B has a meltingtemperature in the range of about 236-238° C., as found by visualdetermination, and exhibits a corresponding endothermic event at about241° C., as shown by the representative trace in FIG. 10. Subsequent tothis endothermic event, tacedinaline Form B undergoes a thermallyactivated dehydrative intramolecular cyclization to formN-(4-(1-H-benzo[d]imidazol-2-yl)acetamide, which exhibits acharacteristic endothermic event at about 310° C.

An exemplary listing of representative XRPD peaks of an embodiment oftacedinaline Form B can be found in Table 3. An exemplary listing ofrepresentative IR peaks of an embodiment of tacedinaline Form B can befound in Table 4.

Crystalline tacedinaline Form B exhibits improved properties relative tothe form of tacedinaline disclosed in the art (Form C). For example,Form B has improved thermal stability relative to tacedinaline Form C.In addition, Form B does not contain methanol, which may be undesirablein various embodiments. Further, the solubility of Form B in water issuperior to that of Forms A and D.

TABLE 3 Form B Degrees 2θ 5.96 7.22 10.94 11.41 11.93 13.40 14.80 17.7518.09 21.53 27.89

TABLE 4 Form B Reciprocal cm 3432.1 3291.1 1668.5 1645.0 1599.6 1527.01506.0 1489.0 1455.7 1404.7 1371.1 1307.6 1294.9 1217.6 1181.3 1154.91122.9 1038.3 1017.3 964.4 905.2 856.2 845.4 827.5 778.1 738.5 693.3674.6 631.6 605.5

Crystalline Tacedinaline Form D

Crystalline tacedinaline Form D was obtained in a crystalline solid formthat is characterized by a unique XRPD pattern substantially as shown inFIG. 26, and an IR spectrum substantially as shown in FIG. 27, the¹H-NMR as shown in FIGS. 39A-39C, and the UPLC/MS as shown in FIGS.40A-40C. Tacedinaline Form D was found to be an anhydrate, as suggestedby the representative TGA plot in FIG. 28 that exhibits no weight lossprior to decomposition. The anhydrous nature of Form D was confirmed bythe single crystal structure. Form D has a melting temperature in therange of about 240-244° C., as found by visual determination, andexhibits a corresponding endothermic event at about 238° C., as shown bythe representative DSC trace in FIG. 29. Subsequent to this endothermicevent, tacedinaline Form D undergoes a thermally activated dehydrativeintramolecular cyclization to formN-(4-(1-H-benzo[d]imidazol-2-yl)acetamide, which exhibits acharacteristic endothermic event at about 310° C.

An exemplary listing of representative XRPD peaks of an embodiment oftacedinaline Form D can be found in Table 5. An exemplary listing ofrepresentative IR peaks of an embodiment of tacedinaline Form D can befound in Table 6.

Crystalline tacedinaline Form D exhibits improved properties relative toother forms of tacedinaline, including that disclosed in the art (FormC). For example, Form D has improved thermal stability relative totacedinaline Form C. In addition, Form D does not contain methanol,which may be undesirable in various embodiments. Further, Form D is morethermodynamically stable under certain conditions relative to Form B, asshown in Examples 10A and 12.

TABLE 5 Form D Degrees 2θ 7.31 11.96 13.97 14.35 14.81 17.87 18.31 21.5422.35 25.34

TABLE 6 Form D Reciprocal cm 3443.4 3447.1 3290.7 1666.4 1645.7 1602.81530.5 1507.4 1488.9 1455.7 1404.8 1371.4 1307.9 1297.7 1282.4 1271.61218.5 1184.0 1155.2 1124.0 1039.1 1017.5 964.8 904.4 858.0 845.7 830.5777.5 759.0 738.2 669.8 605.3

Crystalline Tacedinaline TFA Salt

A novel tacedinaline TEA (trifluroacetate) salt was obtained in acrystalline solid form, characterized by a unique XRPD pattern as shownin FIG. 33. Representative TGA plot can be found in FIG. 35, andrepresentative DSC can be found in FIG. 36. Subsequent to theendothermic event at about 257° C., as can be seen in the DSC trace, itappears that the novel crystalline tacedinaline TFA salt undergoes athermally activated dehydrative intramolecular cyclization to formN-(4-(1-H-benzo[d]imidazol-2-yl)acetamide, which exhibits acharacteristic endothermic event at about 310° C.

An exemplary listing of representative XRPD peaks of an embodiment ofthe crystalline tacedinaline TFA salt can be found in Table 7. Anexemplary listing of representative IR peaks of an embodiment of thecrystalline tacedinaline TFA salt can be found in Table 8.

In at least one embodiment, the use of trifluroacetic acid providesoptimal reaction conditions and yields for the deprotection of the BOCintermediate (see Example 15, below). The isolation of the resultingtrifluroacetate salt allowed for decreased residual TEA, reducedreaction volumes during the neutralization reaction, increased reactionyields, shorter reaction times, and/or higher purity of the resultingfree base.

An exemplary listing of representative XRPD peaks of an embodiment ofcrystalline tacedinaline TFA salt can be found in Table 7. An exemplarylisting of representative IR peaks of an embodiment of crystallinetacedinaline TFA salt can be found in Table 8.

TABLE 7 TFA Salt Degrees 2θ 5.47 11.12 17.54 19.30 20.47

TABLE 8 TFA salt Reciprocal cm 3304.0 3255.8 1777.0 1668.0 1641.0 1600.61498.8 1452.8 1431.0 1407.2 1372.5 1316.7 1268.3 1182.2 1122.1 1037.51020.0 866.8 839.1 797.1 769.0 750.3 717.1 687.4 632.0 606.9 549.2

Amorphous Tacedinaline

Amorphous tacedinaline was obtained in a solid form as a mixture withN-(4-(1-H-benzo[d]imidazol-2-yl)acetamide, and is characterized by anXRPD pattern having a classic “amorphous halo”, as shown in FIG. 30. Theamorphous tacedinaline mixture has a glass transition temperature atabout 82° C., exhibits an endothermic event at about 132° C., and twoendothermic events at about 227° C. and 232° C., as shown by therepresentative DSC trace in FIG. 42.

As can be seen from the differences in XRPD patterns in FIG. 25,crystalline tacedinaline Forms A, B, C, and D, and the novel crystallinetacedinaline TFA salt, each exhibit unique crystallographic properties,and each represents a distinct crystal form of the compound. As can alsobe seen from FIG. 25, the amorphous mixture exhibits a broad,featureless diffraction pattern, as is typical of a non-crystallineform.

The invention in various exemplary embodiments relates to pure orsubstantially pure crystalline tacedinaline Form A, crystallinetacedinaline Form B, crystalline tacedinaline Form D, the novelcrystalline tacedinaline TFA salt form, or amorphous tacedinaline. Byway of example, in one embodiment, the invention may relate to a batchor lot of tacedinaline which is 50% or more, such as 60% or more, 75% ormore, 90% or more, 95% or more, 98% or more, or 99% or more, crystallinetacedinaline Form A, crystalline tacedinaline Form B, crystallinetacedinaline Form D, the novel crystalline tacedinaline TFA salt form,or amorphous tacedinaline. In further exemplary embodiments, theinvention relates to pharmaceutical compositions and/or formulationscomprising pure or substantially pure crystalline tacedinaline Form A,crystalline tacedinaline Form B, crystalline tacedinaline Form D, thenovel crystalline tacedinaline TFA salt form, or amorphous tacedinaline.

In further embodiments, the invention relates to crystallinetacedinaline Form A, crystalline tacedinaline Form B, crystallinetacedinaline Form D, the novel crystalline tacedinaline TFA salt form,and/or amorphous tacedinaline in a mixture, such as a mixture ofcrystalline tacedinaline Forms A, B, and/or D, the novel crystallinetacedinaline TFA salt form, and/or amorphous tacedinaline, or in amixture comprising additional known or as yet unknown solid forms oftacedinaline. By way of example only, the mixture may comprise one ormore of crystalline tacedinaline Forms A, B, and D, the novelcrystalline tacedinaline TFA salt form, and/or amorphous tacedinaline incombination with Form C.

Pharmaceutical Compositions and Methods of Treatment

The novel solid forms of tacedinaline of the invention possesssubstantially the same pharmacological activity as the known form oftacedinaline, and are therefore useful in methods of treating,alleviating, and/or preventing various conditions including, forexample, neoplastic diseases, memory loss, and cognitive functiondisorders/impairments. Neoplastic diseases include, for example, cancerssuch as prostate, breast, colon, and brain, including without limitationglioblastoma.

Exemplary cognitive function disorders that may be treated, alleviated,and/or prevented according to various embodiments of the inventioninclude, but are not limited to, those disclosed in WO 2011/053876 A1,incorporated herein by reference in its entirety. By way of exampleonly, cognitive function disorders/impairments that may be treated,alleviated, and/or prevented include those associated with Alzheimer'sdisease, Huntington's disease, seizure-induced memory loss,schizophrenia, Rubinstein-Taybi syndrome, Rett syndrome, Fragile X, Lewybody dementia, vascular dementia, attention deficit hyperactivitydisorder (ADHD), dyslexia, bipolar disorder, anxiety disorders,conditioned fear response, panic disorders, obsessive compulsivedisorders, post-traumatic stress disorder, phobias, social anxietydisorders, substance dependence recovery, and social, cognitive, andlearning disorders associated with autism, traumatic head injury, orattention deficit disorder (ADD).

By use of the term “treating” or “alleviating” herein, it is meantdecreasing the symptoms, markers, and/or any negative effects of acondition in any appreciable degree in a patient who currently has thecondition. By use of the term “preventing” it is meant preventingentirely or preventing to some extent, such as, for example, byinhibiting all together or delaying the onset or lessening the degree towhich a patient develops a condition.

In further embodiments of the invention are provided methods ofimproving cognitive function in a normal subject. As used herein, a“normal subject” is a subject that has not been diagnosed with adisorder associated with impaired cognitive function. Improvingcognitive function includes promoting cognitive function in a subject sothat the subject more closely resembles or exceeds the function of anage-matched normal, unimpaired subject.

Further exemplary embodiments of the invention relate to methods ofpromoting fear extinction in a subject.

The methods of the invention described herein may be accomplished byadministering to a subject a therapeutically effective amount oftacedinaline comprising crystalline tacedinaline Form A, crystallinetacedinaline Form B, crystalline tacedinaline Form D, the novelcrystalline tacedinaline TFA salt form, and/or amorphous tacedinaline.As used herein, a “therapeutically effective amount” refers to an amountof a therapeutic agent sufficient to treat, alleviate, and/or prevent acondition by administration of a composition of the invention. Thatamount is any amount sufficient to exhibit a detectable therapeuticand/or preventative and/or ameliorative effect, and can be determined byroutine experimentation by those of skill in the art. The effect mayinclude treatment, alleviation, and/or prevention of any of thedisorders or conditions listed herein, for example, as well as symptomsassociated therewith. The actual amount required for treatment of anyparticular patient will depend upon a variety of factors including thedisorder being treated and its severity; the specific pharmaceuticalcomposition employed; the age, body weight, general health, sex and dietof the patient; the mode of administration; the time of administration;the route of administration; and the rate of excretion of tacedinaline;the duration of the treatment; any drugs used in combination with orcoincidental to the treatment; and other such factors well known in themedical arts.

The invention in various exemplary embodiments relates to pharmaceuticalcompositions and formulations comprising crystalline tacedinaline FormA, tacedinaline Form B, tacedinaline Form D, the novel crystallinetacedinaline TFA salt form, and/or amorphous tacedinaline, in anyamount. Thus, for example, the invention in various embodiments relatesto pharmaceutical compositions and formulations comprising even one or afew crystals of tacedinaline Form A, tacedinaline Form B, and/ortacedinaline Form D, the novel crystalline tacedinaline TFA salt form,and/or one or a few particles of amorphous tacedinaline. As a furtherexample, the pharmaceutical compositions and formulations may comprisecrystalline tacedinaline Form A, Form B, and/or Form D, the novelcrystalline tacedinaline TFA salt form, and/or amorphous tacedinaline,in an amount sufficient to be detected by analytical methods known inthe art, such as, for example, IR, XRPD, Raman spectroscopy, and thelike.

In further exemplary embodiments, the invention relates topharmaceutical compositions and formulations comprising atherapeutically effective amount of tacedinaline comprising any amountof crystalline tacedinaline Form A, tacedinaline Form B, and/ortacedinaline Form D, the novel crystalline tacedinaline TFA salt form,and/or amorphous tacedinaline. As such, the amount of any one of or acombination of, crystalline tacedinaline Form A, tacedinaline Form B,and/or tacedinaline Form D, the novel crystalline tacedinaline TFA saltform, and/or amorphous tacedinaline, may not themselves be present in atherapeutically effective amount in various exemplary pharmaceuticalcompositions and formulations. However, as contemplated herein, as longas some amount of at least one of crystalline tacedinaline Forms A, B,and/or D, the novel crystalline tacedinaline TFA salt form, and/oramorphous tacedinaline, is present, the pharmaceutical compositionand/or formulation is within the scope of the invention.

A pharmaceutical composition of the invention may be in anypharmaceutical form which contains any amount of crystallinetacedinaline Forms A, B, and/or D, the novel crystalline tacedinalineTFA salt form, and/or amorphous tacedinaline, as described herein. Forexample, the pharmaceutical compositions of the invention may beformulated in unit dosage form for ease of administration and uniformityof dosage. A “unit dosage form” refers to a physically discrete unit oftherapeutic agent appropriate for the patient to be treated. In oneexemplary embodiment, the pharmaceutical composition of the invention isa solid unit dosage form that maintains the solid form of at least someamount of crystalline tacedinaline Forms A, B, and/or D, the novelcrystalline tacedinaline TFA salt form, and/or amorphous tacedinaline.Unit dosage forms include, but are not limited to, those disclosed in WO2011/053876 A1.

Solid unit dosage forms useful for oral administration according to theinvention include, for example, capsules, tablets, pills, powders, andgranules. The active ingredient may optionally be administered in aformulation that provides quick release, sustained release or delayedrelease after administration to the patient. In such solid unit dosageforms, the active compound may be mixed with at least one inert,pharmaceutically acceptable carrier, such as sodium citrate or dibasiccalcium phosphate, or any other pharmaceutically acceptable carrierknown in the art. The choice of the pharmaceutically acceptable carrierdepends upon the pharmaceutical form and the desired method ofadministration to be used. For a pharmaceutical composition of theinvention, that is one having crystalline tacedinaline Forms A, B,and/or D, the novel crystalline tacedinaline TFA salt form, and/oramorphous tacedinaline, a carrier should be chosen that maintains thesolid form of at least some amount of at least one of crystallinetacedinaline Forms A, B, and/or D, the novel crystalline tacedinalineTFA salt form, and/or the amorphous form. In other words, the carriershould not substantially alter the solid form of the entire quantity ofcrystalline tacedinaline Forms A, B, and/or D, the novel crystallinetacedinaline TFA salt form, and/or amorphous tacedinaline, present. Norshould the carrier be otherwise incompatible with tacedinaline itself,or with crystalline tacedinaline Forms A, B, and D, the novelcrystalline tacedinaline TFA salt form, and/or amorphous tacedinaline,as described herein, such as by producing any undesirable biologicaleffect or otherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutical composition.

The solid unit dosage form may also include one or more other componenttypically used in formulating pharmaceutical dosage forms, as well knownin the art, such as, for example: a) fillers or extenders such asstarches, lactose, sucrose, glucose, mannitol, and silicic acid; b)binders such as, for example, carboxyrnethylcellulose, alginates,gelatin, polyvinylpyrrolidinone, sucrose, and acacia; c) humectants suchas glycerol; d) disintegrating agents such as agar, calcium carbonate,potato or tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; e) dissolution retarding agents such as paraffin; f)absorption accelerators such as quaternary ammonium compounds; g)wetting agents such as, for example, cetyl alcohol and glycerolmonostearate; h) absorbents such as kaolin and bentonite clay; and i)lubricants such as talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, and sodium lauryl sulfate. The solid unit dosageforms may also comprise buffering agents. They may optionally containopacifying agents and can also be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain part of theintestinal tract, optionally, in a delayed manner. Other componentsuseful in the unit dosage forms according to the invention include, butare not limited to, those disclosed in WO 2011/053876 A1. Remington'sPharmaceutical Sciences, Sixteenth Edition, E. W. Martin (MackPublishing Co., Easton, Pa., 1980) discloses various carriers used informulating pharmaceutical techniques for the preparation thereof. Solidunit dosage forms of pharmaceutical compositions of the invention canalso be prepared with coatings and shells such as enteric coatings andother coatings well known in the pharmaceutical formulating art.

Solid unit dosage forms comprising the amorphous form of tacedinalinedescribed herein may also comprise stabilizing excipients. Becausecrystalline forms are often more thermodynamically stable than amorphousforms, there is a driving force toward crystallization of the amorphousstate, and thus a need to stabilize the formulation. Such stabilizingexcipients may include, but are not limited to, polymers, celluloses,and organic acids. Exemplary stabilizing excipients includepolyvinylpyrrolidone (PVP), hydroxypropyl cellulose (HPC),hydroxypropylmethyl cellulose (HPMC), hydroxypropyl methacrylamide(HMPA), polyethylene glycols (PEGs), and citric acid, to name a few.

It is well known that pharmaceutical excipients, such as carriers,fillers, binders, and the like, typically found in solid unit dosageforms, may make detection of a particular solid form of a compound, suchas crystalline tacedinaline Forms A, B, and/or D, the novel crystallinetacedinaline TFA salt form, and/or amorphous tacedinaline, difficult.This may be due, for example, to interference in analytical techniquessuch as XRPD or IR. However, one of skill in the art, using techniquesknown in the art, should generally be able to determine whether aparticular solid form is present.

Any of crystalline tacedinaline Forms A, B, and/or D, the novelcrystalline tacedinaline TFA salt form, and/or amorphous tacedinaline,may also be used in, or used in preparation of, non-solid formulations,such as, for example, a solution, an injectable or inhalableformulation, or a patch. Such non-solid formulations are known in theart. In a non-solid formulation, the crystalline and/or amorphous formmay, in various embodiments, not be maintained. For example, thecrystalline and/or amorphous form may be dissolved in a liquid carrier.The crystalline and/or amorphous forms of the invention may provideadvantages of handling stability and purity to the process of makingsuch formulations.

In a further exemplary embodiment, the crystalline tacedinaline Forms A,B, and/or D, the novel crystalline tacedinaline TFA salt form, and/oramorphous tacedinaline, may be administered in a suspension.

In addition, any of crystalline tacedinaline Forms A, B, and/or D, thenovel crystalline tacedinaline TFA salt form, and/or amorphoustacedinaline may be used as a starting material or intermediate in aprocess of preparing a different solid form of tacedinaline, or byconverting one solid form to another. Additionally, in furtherembodiments contemplated herein, any of crystalline tacedinaline FormsA, B, and/or D, the novel crystalline tacedinaline TFA salt form, and/oramorphous tacedinaline, may be used in the preparation of solidformulations that do or do not ultimately contain any of crystallinetacedinaline Forms A, B, and/or D, the novel crystalline tacedinalineTFA salt form, and/or amorphous tacedinaline (for example, by conversionof one or more of crystalline tacedinaline Forms A, B, and/or D, thenovel crystalline tacedinaline TFA salt form, and/or amorphoustacedinaline, to some other solid form). As used herein, the term“converting” with regard to converting one form to another is intendedto include any step or condition that changes the solid form of thecompound, such as, for example, a process that uses a particular form asan intermediate; a formulating step that causes intentional orunintended conversion, such as direct compression or wet granulation;exposure to heat and/or humidity; etc. By way of example only, thecrystalline tacedinaline TFA salt form described herein may be used in aprocess for preparing crystalline tacedinaline Forms A and/or B, forexample as an intermediate product.

The invention in various embodiments also relates to the treatment,prevention, and/or alleviation of neoplastic diseases, memory loss, andcognitive function disorders/impairments, comprising administering to asubject crystalline tacedinaline Forms A, B, and/or D, the novelcrystalline tacedinaline TFA salt form, and/or amorphous tacedinaline,or a pharmaceutical composition comprising crystalline tacedinalineForms A, B, and/or D, the novel crystalline tacedinaline TFA salt form,and/or amorphous tacedinaline. In further embodiments, methods ofimproving cognitive function in a normal subject and/or methods ofpromoting fear extinction in a subject comprising administering to asubject crystalline tacedinaline Forms A, B, and/or D, the novelcrystalline tacedinaline TFA salt form, and/or amorphous tacedinaline,or a pharmaceutical composition comprising crystalline tacedinalineForms A, B, and/or D, the novel crystalline tacedinaline TFA salt form,and/or amorphous tacedinaline, are disclosed. In various embodiments, apharmaceutical composition administered comprises an effective amount oftacedinaline comprising crystalline tacedinaline Forms A, B, and/or D,the novel crystalline tacedinaline TFA salt form, and/or amorphoustacedinaline. These solid forms and pharmaceutical compositionscontaining them may, according to various embodiments, be administeredusing any amount, any form of pharmaceutical composition, and any routeof administration effective for the desired treatment.

The crystalline tacedinaline Forms A, B, and/or D, the novel crystallinetacedinaline TFA salt form, and/or amorphous tacedinaline, according tothe invention may be administered by any route known, such as, forexample, orally, transdermally, intravenously, cutaneously,subcutaneously, nasally, intramuscularly, intraperitoneally,intracranially, and intracerebroventricularly.

In certain exemplary embodiments, the tacedinaline comprisingcrystalline tacedinaline Forms A, B, and/or D, the novel crystallinetacedinaline TFA salt form, and/or amorphous tacedinaline, may beadministered at dosage levels of greater than about 0.001 mg/kg, such asgreater than about 0.01 mg/kg or greater than about 0.1 mg/kg. Forexample, the dosage level may be from about 0.001 mg/kg to about 50mg/kg, such as from about 0.01 mg/kg to about 25 mg/kg, from about 0.1mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 5 mg/kg ofsubject body weight per day, one or more times a day, to obtain thedesired therapeutic effect. It will also be appreciated that dosagessmaller than 0.001 mg/kg or greater than 50 mg/kg (for example 50-100mg/kg) can also be administered to a subject. In one exemplaryembodiment, a dosage of up to about 0.4 mg/kg, once a day for at least 2consecutive days, such as for 14 consecutive days, may be administered.

In another exemplary embodiment, administration could be on anintermittent schedule. By way of example only, a dosage of up to about0.1 mg/kg once a day for up to 56 consecutive days may be administeredfollowed by a dosing holiday, and then an additional dosing schedule.

Further, administration less frequently than daily, such as, forexample, every other day, may be chosen. In additional exemplaryembodiments, administration with at least 2 days between doses may bechosen. By way of example only, dosing may be every third day,bi-weekly, or weekly. For example, a dosage of up to about 0.8 mg/kgevery other day, may be given. As another example, a single, acute dosemay be administered. By way of example a one-time dose of up to about 50mg/kg may be administered, such as about 10 mg/kg, or about 2.2 mg/kg.

As discussed above, the amount required for treatment of a particularpatient will depend upon a variety of factors well known in the medicalarts, and may vary depending on, for example, the condition beingtreated. And, as also discussed, the pharmaceutical compositioncomprising the crystalline tacedinaline Forms A, B, and/or D, the novelcrystalline tacedinaline TFA salt form, and/or amorphous tacedinaline,may be administered as a unit dosage form.

In further embodiments, the subject to which the crystallinetacedinaline Forms A, B, and/or D, the novel crystalline tacedinalineTFA salt form, and/or amorphous tacedinaline, is administered mayundergo additional therapies in combination therewith. The combinationtherapies may be any therapy appropriate for the disease or disorderbeing treated. For example, the combination therapy may includebehavioral therapy and/or additional pharmaceutical compounds. Exemplarybehavioral therapies and additional pharmaceutical compounds that may beuseful in combination therapies contemplated herein may include, but arenot limited to, those disclosed in WO 2011/1053876 A1.

In various embodiments, the pharmaceutical compositions or formulationscomprising crystalline tacedinaline Forms A, B, and/or D, the novelcrystalline tacedinaline TFA salt form, and/or amorphous tacedinaline,may be assembled into therapeutic, diagnostic, or research kits tofacilitate their use in a particular application. Such a kit maycomprise, for example, a housing, an effective amount of tacedinalinecomprising crystalline tacedinaline Forms A, B, and/or D, the novelcrystalline tacedinaline TFA salt form, and/or amorphous tacedinaline,formulated for oral, transdermal, intraveneous, cutaneous, subcutaneous,nasal, intramuscular, intraperitoneal, intracranial, andintracerebroventricular administration, and instructions foradministering the tacedinaline to a subject in need thereof. Exemplarykits that may be useful include, but are not limited to, those disclosedin WO 2011/053876 A1.

Although the present invention herein has been described with referenceto various exemplary embodiments, it is to be understood that theseembodiments are merely illustrative of the principles and applicationsof the present invention. Those having skill in the art would recognizethat a variety of modifications to the exemplary embodiments may bemade, without departing from the scope of the invention.

The use of the terms “the,” “a,” “an,” or other singular terms, is meantto include plural embodiments as well, and vice versa. In addition, itshould be understood that all numbers herein are modified by “about,”whether or not so stated.

Moreover, it should be understood that various features and/orcharacteristics of differing embodiments herein may be combined with oneanother. It is therefore to be understood that numerous modificationsmay be made to the illustrative embodiments without departing from thescope of the invention.

Furthermore, other embodiments of the invention will be apparent tothose skilled in the art from consideration of the specification andpractice of the invention disclosed herein. It is intended that thespecification and examples be considered as exemplary only, with a scopeand spirit being indicated by the claims.

EXAMPLES Example 1 Preparation and Characterization of a Mixture ofCrystalline Tacedinaline Forms B and D

2-Nitroaniline 1.1 (10.0 g, 72.4 mmol, 1.0 eq.) was dissolved in dry DMF(100 mL) and cooled to 0° C. Subsequently, 60% NaH (1.91 g, 80.0 mmol,1.1 eq.) was added slowly to the reaction mixture under an argonatmosphere. After 30 minutes, di-tert-butyl dicarbonate (17.3 g, 80.0mmol, 1.1 eq.) dissolved in dry DMF (50 mL) was added slowly to thereaction mixture at that temperature. The reaction mixture was slowlybrought to 23° C. and further stirred for 5 hours. After completion, thereaction mixture was poured into ice water and the precipitated solidwas filtered, washed with water (3×100 mL), and dried. The material wasdissolved in EtOAc/hexanes and passed through short silica gel column.The filtrate was concentrated in vacuo to provide tert-butyl-2-nitrophenylcarbamate 1.2 as a pale yellow solid. Yield 1.2=9.3 g (54%).

¹H NMR (500 Hz, d⁶-DMSO) δ 9.58 (s, 1H), 7.95 (d, J=7.5 Hz, 1H), 7.66(app d, J=3.5 Hz, 2H), 7.32-7.26 (m, 1H), 1.45 (s, 9H)

tert-Butyl-2-nitrophenylcarbamate 1.2 (9.0 g, 37.8 mmol, 1.0 eq.), iron(III) chloride (0.4 g, 2.3 mmol, 0.06 eq.), hydrazine monohydrate (51.0g, 1592 mmol, 42 eq.) and Maki (150 mL) were combined and heated to 90°C. After vigorously stirring for 2-3 hours, the reaction mixture wasfiltered hot through celite and washed with EtOAc. The solvents wereremoved under reduced pressure. This crude residue was diluted with coldwater. The resultant solid was filtered and washed with hexane to affordtert-butyl 2-aminophenyl carbamate 1.3 as an off white solid. Yield1.3=7 g (90%).

¹H NMR (500 Hz, d⁶-DMSO) δ 8.25 (s, 1H), 7.16 (d, J=7.5 Hz, 1H), 6.83(t, J=7.5 Hz, 1H), 6.66 (d, 1=7.5 Hz, 1H), 6.52 (t, J=7.5 Hz, 1H), 4.80(s, 2H), 1.45 (s, 9H); MS (ESI+): m/z 231 [M+Na]⁺.

tert-Butyl 2-aminophenyl carbamate 1.3 (3.0 g, 14.4 mmol, 1 eq.),4-acetamido benzoic acid 1.4 (2.8 g, 15.8 mmol, 1.1 equiv.), and(benzotriazol-1-yloxy)tris (dimethylamino)phosphoniumhexafluorophosphate (7.6 g, 17.3 mmol, 1.2 eq.) were dissolved inpyridine (20 mL). After stirring at 23° C. for 48 hours, the reactionmixture was added to water and stirred. The resultant precipitated solidwas filtered, washed with water, washed with ether, and dried undervacuum to provide tert-butyl (2-(4-acetamidobenzamido)phenyl)carbamate1.5. Yield 1.5=4.8 g (90%).

¹H NMR (500 Hz, d⁶-DMSO) δ 10.23 (s, 1H), 9.73 (bs, 1H), 8.66 (bs, 1H),7.90 (d, J=8.5 Hz, 2H), 7.72 (d, =9 Hz, 2H), 7.53 (t, 0.1=8.5 Hz, 2H),7.22-7.12 (m, 2H), 2.09 (s, 3H), 1.45 (s, 9H); MS (ESI+): m/z 392[M+Na]⁺.

To a 0° C. solution of tert-butyl (2-(4-acetamidobenzamido)phenyl)carbamate 1.5 (3.5 g, 9.5 mmol) in dry dichloromethane (55 mL) was addedtrifluoroacetic acid (22 mL) dropwise. The mixture was allowed to slowlywarm to 23° C. for 2 hours until the reaction was complete. The solventswere removed in vacuo. The reaction mixture was diluted with water andthe pH was adjusted to ˜8 with a saturated aqueous solution of sodiumbicarbonate. The resulting precipitate was filtered, washed with waterand ether, and dried under vacuum to afford4-acetamido-N-(2-aminophenyl)benzamide 1.6 as an off-white solid. Yield1.6=2.4 g (96%).

¹H NMR (500 Hz, d⁶-DMSO) δ 10.18 (s, 1H), 9.54 (s, 1H), 7.93 (d, =8.5Hz, 2H), 7.69 (d, J 8.5 Hz, 2H), 7.15 (d, J=7.5 Hz, 1H), 6.96 (t, J=7.5Hz, 1H), 6.78 (d, J=7, 5 Hz, 1H), 6.59 (t, 7.5 Hz, 1H), 4.88 (s, 2H),2.08 (s, 3H). MS (ESI+): m/z 269.9 [M+H]⁺.

Analytical data were obtained on the final product 1.6: the XRPD patternwas as shown in FIG. 13, the JR spectrum was substantially as shown inFIG. 14, the TGA profile was substantially as shown in FIG. 15, and theDSC trace was substantially as shown in FIG. 16.

The analytical data obtained on product 1.6 indicate that it is amixture of crystalline tacedinaline Forms B and D.

Example 2 Preparation and Characterization of Crystalline TacedinalineForm A

A mixture of 202.3 mg of the mixture of crystalline tacedinaline Forms Band D (1.6) from Example 1 and 15 mL, of a 2:1 (volume:volume) solutionof ethanol:water was brought to gentle reflux. Most of the soliddissolved. The mixture was filtered hot through filter paper. Thefiltrate, from which some solid had separated during the filtration, wasreheated to gentle reflux and additional ethanol:water solution wasadded to bring the total volume back to about 15 mL. After all of thesolid had dissolved, the mixture was removed from the hot plate,covered, and allowed to cool to ambient temperature. Crystals formed.The mixture was placed in the refrigerator for about 15 minutes and thenfiltered to give 125.6 mg of crystalline solid 2.1. A portion of thatsample was ground using an agate mortar and pestle to give the finalproduct, 2.2.

Analytical data were obtained on the final product 2.2: the XRPD patternwas as shown in FIG. 1A, the IR spectrum was as shown in FIG. 2, the TGAprofile was as shown in FIG. 3, the DSC trace was as shown in FIG. 4,the ¹H-NMR spectrum was as shown in FIGS. 5A-5C, and the LC/MS data wasas shown in FIG. 6.

The analytical data obtained on product 2.2 indicate that it iscrystalline tacedinaline Form A.

Example 3 Single Crystal X-Ray Analysis of Crystalline Tacedinaline FormA

A single crystal suitable for x-ray diffraction analysis was selectedfrom the product 2.1 of Example 2, above.

The crystallographic data collection and single crystal parameters forthe tacedinaline Form A crystal are set forth in Table 9.

TABLE 9 formula C₁₅H₁₅N₃O₂ formula weight 269.31 space group P 1 21/c 1(No. 14) a (Å) 6.5031(3) b (Å) 9.0907(4) c (Å) 22.6701(16) β (degrees)95.010(7) volume (Å³) 1335.08(13) Z 4 d_(calc) (g cm⁻³) 1.340 crystaldimensions (mm) 0.18 × 0.18 × 0.05 temperature (K) 150 radiation(wavelength in Å) Cu Ka (1.54184) monochromator graphite linear abs coef(mm−1) 0.706 absorption correction applied empirical^(a) transmissionfactors (min, max) 0.83, 0.97 diffractometer Nonius Kappa CCD h, k, lrange −7 to 7 −10 to 10 −25 to 26 2θ range (deg) 13.67-132.87 mosaicity(deg) 2.97 programs used SHELXTL F₀₀₀ 568.0 weighting 1/[σ²(Fo²) +(0.0882P)² + 0.0000P] where P = (Fo² + 2Fc²)/3 data collected 14576unique data 1973 R_(int) 0.063 data used in refinement 1973 cutoff usedin R-factor calculations F_(o) ² > 2.0 s(F_(o) ²) data with I > 2.0 s(I)1201 refined extinction coef 0.0049 number of variables 199 largestshift/esd in final cycle 0.00 R(F_(o)) 0.060 Rw(F_(o) ²) 0.138 goodnessof fit 1.059 ^(a)Sheldrick, G. M., SADABS 1996, Gottingen, Germany.

Example 4 Preparation and Characterization of Crystalline TacedinalineForm A

A slurry of 50.7 mg of the mixture of crystalline tacedinaline Forms Band D (1.6) from Example 1 and about 1 mL of a 4:1 (volume:volume)solution of acetonitrile:water was stirred at ambient temperatureovernight. Filtration and vacuum drying of the filter cake (ambienttemperature, diaphragm pump pressure, 1 hour) yielded 40.4 mg of white,crystalline solid.

Analytical data were obtained on the product: the XRPD pattern was asshown in FIG. 1B, the IR spectrum was substantially as shown in FIG. 2,the TGA profile was substantially as shown in FIG. 3, and the DSC tracewas substantially as shown in FIG. 4.

The analytical data obtained on the product indicate that it iscrystalline tacedinaline Form A.

Example 5A Preparation and Characterization of Crystalline TacedinalineForm B

A mixture of 100.5 mg of a mixture of crystalline tacedinaline Forms Band D (1.6) from Example 1 and 30 mL, of acetone was brought to gentlereflux. All solids dissolved. The solution was filtered hot throughfilter paper. The clear filtrate was brought to gentle reflux and thevolume reduced to about 10 mL by boiling. IR was allowed to stand atroom temperature overnight, then in the refrigerator and freezer for ashort time. No crystallization occurred, so the solution was againbrought to gentle reflux and reduced in volume to about 3 mL by boiling.It was allowed to cool to ambient temperature and crystallizationoccurred. After about one hour at ambient temperature it was placed inthe refrigerator for about 30 minutes and filtered to give 14.3 mg ofwhite, crystalline solid.

Analytical data were obtained on the product: the XRPD pattern was asshown in FIG. 7B, the IR spectrum was as shown in FIG. 8, the TGAprofile was as shown in FIG. 9, the DSC trace was as shown in FIG. 10,the ¹H-NMR spectrum was as shown in FIGS. 11A-11C, and the LC/MS datawas as shown in FIG. 12.

The analytical data obtained on the product indicate that it iscrystalline tacedinaline Form B.

Example 5B Preparation and Characterization of Crystalline TacedinalineForm B

A mixture of 204.0 mg of crystalline tacedinaline, which is a mixture ofForms B and D, and 15 mL of a 2:1 (volume:volume) solution ofTHF:ethanol was brought to gentle reflux. Most of the solid dissolved.The mixture was filtered hot through hardened filter paper and thefilter cake was retained. The clear filtrate was reheated to gentlereflux and reduced in volume by boiling. Crystallization occurred at avolume of about 8 mL. When the volume was about 5 mL, the mixture wasremoved from the hot plate and allowed to cool to ambient temperature.It was then covered and placed in the refrigerator overnight. Filtrationand drying of the filter cake (ambient temperature, diaphragm pumppressure, 90 minutes) gave 121.6 mg of crystalline solid.

Analytical data were obtained on the product: the XRPD pattern wassubstantially as shown in FIG. 7B, and the DSC trace was substantiallyas shown in FIG. 10.

The analytical data obtained on the product indicate that it iscrystalline tacedinaline Form B.

Example 6 Preparation and Characterization of Crystalline TacedinalineForm B

A mixture of 74.5 mg of the mixture of crystalline tacedinaline Form Band Form D (1.6) from Example 1 and 8 mL of acetone was heated to gentlereflux on a hot plate. After about 20 minutes, all solids had dissolved.The solution was filtered through a glass wool plug and again heated togentle reflux on a hot plate in order to concentrate it. When the volumewas about 6 mL the sample was removed from the hot plate and wasobserved to become cloudy as it cooled. The mixture was warmed againuntil it was clear, removed from the hot plate, seeded with a smallamount of crystalline tacedinaline Form B, and placed in therefrigerator overnight. Vacuum filtration and drying of the filter cakeunder diaphragm pump pressure fir about 15 minutes afforded 28.7 mg ofcrystalline product.

Analytical data were obtained on the product: the XRPD pattern was asshown in FIG. 7A, the IR spectrum was substantially as shown in FIG. 8,the TGA profile was substantially as shown in FIG. 9, and the DSC tracewas substantially as shown in FIG. 10.

The analytical data obtained on the product indicate that it iscrystalline tacedinaline Form B.

Example 7 Preparation and Characterization of Crystalline TacedinalineForm C

To the extent possible, the procedure set forth in Thomas, M. et al.,Bioorganic & Medicinal Chemistry 2008, 16, 8109-8116, was followed asdescribed herein. To a stirred solution of 4-acetamido benzoic acid 7.4(2.5 g, 13.95 mmol, 1.0 eq.) in N,N-dimethylformamide (50 mL) was addedo-phenyldiamine 7.7 (4.53 g, 41.9 mmol, 3.0 eq.), followed byN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (3.48 g,18.14 mmol, 1.3 eq.) and catalytic 4-(dimethylamino)pyridine (0.18 g,1.47 mmol, 0.1 eq.). The reaction mixture was stirred at roomtemperature for 16 hours. The solvents were removed under reducedpressure at 58° C. The crude residue was diluted with dichloromethane(100 mL) and kept in the refrigerator overnight. The resultingprecipitate was filtered, washed with hot dichloromethane (100 mL), anddried under vacuum to afford 4-acetamido-N-(2-aminophenyl)benzamide 7.6as an off-white solid. Yield 7.6=2.48 g (66%).

XRPD data was obtained and the crude product was determined to be amixture of Forms B and D. The visual melting point was obtained and wasabout 236° C., although it is noted that the procedure in the abovementioned paper reports a melting point of about 216° C.

In a manner similar to that described in WO 2009/076234, paragraph [64],the crude product 7.6 (0.5 g) was dissolved in 50 mL of hot (70° C.)MeOH:THF (1:1 v/v). The solution was stored at room temperature for 24hours. The resulting white crystals were filtered to provide 0.27 g ofcrystalline product.

Analytical data were obtained on the product: the XRPD pattern was asshown in FIG. 19, the IR spectrum was as shown in FIG. 20, the TGAprofile was as shown in FIG. 21, the DSC trace was as shown in FIG. 22,the ¹H-NMR spectrum was as shown in FIGS. 23A-23C, and the LC/MS datawas as shown in FIG. 24.

The analytical data obtained on the product indicate that it iscrystalline tacedinaline Form C, a methanol solvate.

Example 8 Single Crystal X-Ray Analysis of Crystalline Tacedinaline FormC

A single crystal of tacedinaline Form C suitable for x-ray diffractionanalysis was analyzed. The crystallographic data collection and singlecrystal parameters for the tacedinaline Form C crystal are set forth inTable 10.

TABLE 10 formula C₁₆H₁₉N₃O₃ formula weight 301.35 space group P 1 21/c 1(No. 14) a (Å) 9.7236(5) b (Å) 7.4334(5) c (Å) 20.5398(10) β (degrees)90.133(4) volume (Å³) 1484.60(14) Z 4 d_(calc) (g cm⁻³) 1.348 crystaldimensions (mm) 0.25 × 0.20 × 0.20 temperature (K) 150 radiation(wavelength in Å) Cu Ka (1.54184) monochromator graphite linear abs coef(mm−1) 0.737 absorption correction applied empirical^(a) transmissionfactors (min, max) 0.76, 0.86 diffractometer Nonius Kappa CCD h, k, lrange −11 to 11 −8 to 0 −22 to 24 2θ range (deg) 4.30-144.17 mosaicity(deg) 0.79 programs used SHELXTL F₀₀₀ 640.0 weighting 1/[σ²(Fo²) +(0.0695P)² + 0.7118P] where P = (Fo² + 2Fc²)/3 data collected 6351unique data 2551 R_(int) 0.051 data used in refinement 2551 cutoff usedin R-factor calculations F_(o) ² > 2.0 s(F_(o) ²) data with I > 2.0 s(I)2461 number of variables 221 largest shift/esd in final cycle 0.00R(F_(o)) 0.050 Rw(F_(o) ²) 0.133 goodness of fit 1.079 ^(a)Otwinowski,Z.; Minor, W. Methods Enzymol. 1997, 276, 307

Example 9 Preparation and Characterization of a Mixture of CrystallineTacedinaline Forms B and D

A sample of 506.9 mg of the mixture of crystalline tacedinaline Form Band Form D (1.6) from Example 1 was placed in a vial and treated dropwise with DMF, with warming on a hot plate, until a solution resulted.About 1.5 mL of DMF were required. The solution was added to 10 mL ofcooled dichloromethane. The resulting slurry was placed in the freezerfor about 10 minutes and vacuum filtered. The filter cake was placed ina dessicator under diaphragm pump pressure for about 30 minutes to give424.7 mg (84% yield) of crystalline product.

Analytical data were obtained on the product: the XRPD pattern wassubstantially as shown in FIG. 12, the IR spectrum was as shown in FIG.13, the TGA profile was as shown in FIG. 15, the DSC trace was as shownin FIG. 16, the ¹H-NMR spectrum was as shown in FIGS. 17A-17C, and theLC/MS data was as shown in FIG. 18.

The analytical data obtained on the product indicate that it is amixture of crystalline tacedinaline Forms B and D.

Example 10A Preparation and Characterization of Crystalline TacedinalineForm D

To a mixture of 161.3 mg of crystalline tacedinaline Forms B and D, wasadded 2 mL of acetone. The resulting slurry was stirred at ambienttemperature for 7 days and filtered to give white solid.

Analytical data were obtained on the product: the XRPD pattern was asshown in FIG. 26, the IR spectrum was as shown in FIG. 27, the TGAprofile was as shown in FIG. 28, the DSC trace was as shown in FIG. 29,the ¹H-NMR spectrum was as shown in FIGS. 39A-39C, and the LC/MS datawas as shown in FIGS. 40A-40C.

The analytical data obtained on the product indicate that it iscrystalline tacedinaline Form D.

Example 10B Single Crystal X-Ray Analysis of Crystalline TacedinalineForm D

A slurry of 113.6 mg of a mixture of crystalline tacedinaline Forms Band D was mixed in about 2 mL of a 1:1 (v:v) mixture of acetone:waterand heated on a hot plate with stirring until it refluxed gently.Additional portions of a 1:1 (v:v) mixture of acetone:water were addeduntil all the solid dissolved. About 8 mL total were required. Thesolution was filtered through a glass wool plug and the filtrate wasallowed to stand at ambient temperature in a closed vial overnight,during which time crystallization occurred. The mixture was vacuumfiltered and the crystals were dried under diaphragm pump pressure forabout 15 minutes.

A single crystal of tacedinaline Form D suitable for x-ray diffractionanalysis was selected and analyzed. The crystallographic data collectionand single crystal parameters for the tacedinaline Form D crystal areset forth in Table 11.

TABLE 11 formula C₁₅H₁₅N₃O₂ formula weight 269.31 space group P-1 (No.2) a (Å) 7.2687(5) b (Å) 12.6881(9) c (Å) 15.5597(14) α (degrees)105.784(6) β (degrees) 100.001(6) γ (degrees) 97.274(5) volume (Å³)1336.84(18) Z 4 d_(calc) (g cm⁻³) 1.338 crystal dimensions (mm) 0.24 ×0.12 × 0.04 temperature (K) 150 radiation (wavelength in Å) Cu Ka(1.54184) monochromator graphite linear abs coef (mm−1) 0.705 absorptioncorrection applied empirical^(a) transmission factors (min, max) 0.83,0.97 diffractometer Nonius Kappa CCD h, k, l range 0 to 8, −15 to 15,−18 to 18 2θ range (deg) 6.05-140.29 mosaicity (deg) 0.41 programs usedSHELXTL F₀₀₀ 568.0 weighting 1/[σ²(Fo²) + (0.0610P)² + 0.5417P] where P= (Fo² + 2Fc²)/3 data collected 19661 unique data 4158 R_(int) 0.032data used in refinement 4158 cutoff used in R-factor calculations F_(o)² > 2.0 s(F_(o) ²) data with I > 2.0 s(I) 3411 number of variables 395largest shift/esd in final cycle 0.00 R(F_(o)) 0.043 Rw(F_(o) ²) 0.111goodness of fit 1.056 ^(a)Otwinowski, Z.; Minor, W. Methods Enzymol.1997, 276, 307

Example 11 Competitive Slurry Experiment

A slurry containing 25.8 mg tacedinaline Form A, 25.8 mg of a mixture oftacedinaline Forms B and D, a few milligrams of tacedinaline Form B, and0.5 mL of tetrahydrofuran (THF) was stirred overnight and filtered togive 38.6 mg of a white solid. The white solid was analyzed by XRPD anddetermined to be tacedinaline Form A.

The result of this experiment suggest that crystalline tacedinaline FormA is more thermodynamically stable, relative to tacedinaline Forms B,and D.

Example 12 Solubility Study

Analysis of the relative solubilities of crystalline tacedinaline FormsA, B, and D was performed as follows.

A calibration curve (FIG. 43) was prepared using 5 samples (samples02-06) of various concentrations in water, as set forth in Table 12. Thestarting material was Form B. The only peak in the spectrum, atapproximately 267 nm, was used. The UV parameters were: scan range:190-800 nm; 480 mm/min; 1 cm cuvette.

TABLE 12 Concentration UV absorbance Peak Sample (mg/mL) value (nm) 020.0196 1.44 267.83 03 0.01568 1.16 267.71 04 0.01176 0.87 267.67 050.00588 0.54 267.23 06 0.00196 0.19 267.74

Each sample was placed in water to give a slurry. The slurries werestirred at ambient temperature for 24 hours. The temperature in thelaboratory was monitored and found to be steady at 23° C. After 24hours, the vials containing the slurries were centrifuged for about 5minutes. The mother liquors were decanted and filtered through 0.2micron filters, then diluted appropriately and analyzed by UV. Solidswere dried under dry air purges and analyzed by XRPD. The results areset forth below in Table 13.

TABLE 13 Starting Form Ending Form Solubility (mg/mL) A A 0.0176 B B0.0426 B + D D 0.0208

The results of Examples 10A, 11, and 12 show that relative thermodynamicstability of crystalline tacedinaline Forms A, B, and D is as follows:A>D>B.

Example 13 Accelerated Stability Studies

Accelerated stability studies were performed on samples of crystallinetacedinaline Forms A, B, C, and a mixture of Forms B and D, underconditions of 40° C. and 75% relative humidity. For the studies on FormA, samples were taken at 8 days, 15 days, one month, 10 weeks, and 8months. For the studies on Forms B, C, and a mixture of Forms B and D,samples were taken at 2, 4, 6, 8, and 10 weeks.

The results of the studies on Forms B, C, and a mixture of Forms B andD, which are set forth below in Table 14, show that, at least underthese conditions, crystalline tacedinaline Form B is more stable thanForm C, as Form C converted to either Form D, or a mixture of Form Dwith some amount of Form A present.

As can also be seen from the results Table 14, the mixture of Form B andForm D is also more stable than Form C under these conditions.

TABLE 14 Starting Material Pull Time XRPD Result B 2 weeks B 4 weeks B 6weeks B 8 weeks B C 2 weeks D + A 4 weeks D 6 weeks D + A 8 weeks D + AB + D 2 weeks B + D 4 weeks B + D 6 weeks B + D 8 weeks B + D

The results of the studies on Form A, which are set forth in Table 15below, show that, at least under these conditions, crystallinetacedinaline Form A is stable, as no conversion was seen.

TABLE 15 Starting Material Pull Time XRPD Result A 8 days  A 15 days   A 1 month A 10 weeks A  8 months A

Example 14 Preparation of Amorphous Tacedinaline

Into a platinum TG pan was added 16.7 mg Form A. The sample was heatedin the TG furnace at 10° C./min up to 245° C. The sample was held at245° C. for 2 minutes. Pan was removed from TG furnace and solid wasscraped out. Solid appeared to be a clear glass. Sample was analyzed byXRPD and appears amorphous.

Analytical data were obtained on the product: the XRPD pattern was asshown in FIG. 30, the ¹H-NMR spectrum was as shown in FIG. 31, and theLC/MS data was as shown in FIG. 32.

The analytical data obtained on the product indicate that it is amixture of amorphous tacedinaline andN-(4-(1-H-benzo[d]imidazol-2-yl)acetamide.

Example 15 Synthesis of Crystalline Tacedinaline Form A Synthesis oftert-butyl(2-aminophenyl)carbamate

To a 1 L round-bottom flask, o-phenylenediamine (50.8 g, 0.47 mol) andTHF (500 mL) were added under N₂. To a 500 mL flask, (Boc)₂O (102.5 g,0.47 mol) and THF (150 mL) were added under N₂ and stirred to dissolve.The (Boc)₂O solution was transferred to an addition funnel and addeddropwise into the diamine solution. The reaction mixture was stirred for18 hours at room temperature. A total of 620 mL THF was removed from thereaction mixture under reduced pressure. EtOAc (50 mL) and heptane (400mL) were added, and the resultant slurry was stirred for 1 hour at roomtemperature. The solid was filtered and washed with heptane (2×50 mL).Drying in vacuo at 38° C. for 4 hours afforded the product tert-butyl(2-aminophenyl)carbamate (69.3 g, 71%, 97.3% HPLC purity) as a whitesolid.

¹HNMR (400 Hz, d⁶-DMSO) δ 8.29 (s, 1H), 7.17 (d, I=7.6 Hz, 1 H), 6.83(app dt, J=1.2. Hz, 7.6 Hz, 1H), 6.68 (dd, J=1.2 Hz, 8 Hz, 1H), 6.52(app dt, J=1.2 Hz, 7.6 Hz, 1H), 4.83 (s, 2H), 1.46 (s, 9H).

Synthesis of tert-butyl(2-(4-acetamidobenzamido)phenyl)carbamate

To a 1 L round-bottom flask, were added 4-acetamidobenzoic acid (21.7 g,122 mmol), tert-butyl (2-aminophenyl)carbamate (23.0 g, 111 mmol), andDMF (90 mL). The mixture was stirred to dissolve under N₂, before EDC(27.6 g, 144 mmol) was added in one portion. The reaction mixture wasstirred for 2 h, and water (270 mL) was added and stirred tier 2 h. Theprecipitates were filtered, washed with water (2×45 mL) and heptane(2×45 mL). The filter cake was dried at 40° C. in vacuo for 16 h toafford tert-butyl (2-(4-acetamidobenzamido) phenyl)carbamate (31.8 g,78% yield, 90.6% HPLC purity) as a white solid.

¹HNMR (400 Hz, d⁶-DMSO) δ: 10.27 (s, 1H), 9.76 (s, 1H), 8.70 (bs, 1H),7.91 (d, J=8.8 Hz, 2H), 7.73 (d, J=8.8 Hz, 2H), 7.55-7.51 (m, 2H),7.22-7.13 (m, 2H), 2.10 (s, 3H), 1.45 (s, 9H).

Synthesis of TFA Salt: 4-acetamido-N-(2-aminophenyl)benzamidetrifluoroacetate salt

To a 1 L round-bottom flask, were addedtert-butyl(2-(4-acetamidobenzamido) phenyl)carbamate (31.8 g, 86.2mmol), DCM (1.60 mL) and TFA (95 mL). The reaction mixture was stirredfor 2 hours before concentrating in vacuo. EtOH (95 mL) was added andconcentrated in vacuo to further reduce the amount of residual TFA. MTBE(320 mL) was added, and the suspension was stirred for 2 hours. Thesolids were filtered, washed with MTBE (2×60 mL), and dried at 40° C. invacuo for 18 hours to get 4-acetamido-N-(2-aminophenyl)benzamidetrifluoroacetate salt (32.6 g, 99% yield, 92.2% HPLC purity).

¹HNMR (400 Hz, d⁶-DMSO) δ: 10.28 (s, 1H), 10.07 (s, 2H), 7.98 (d, =8.8Hz, 2 H), 7.73 (d, J=8.4 Hz, 2H), 7.34 (dd, J=1.2 Hz, 8.0 Hz, 1H),7.24-7.14 (m, 2H), 7.09 (t, J=8.0 Hz, 1H), 2.10 (s, 3H). MS (ESI+): m/z271.2 [M+H]⁺.

Synthesis of Crystalline Tacedinaline Free Base Form B

The dried solid of 4-acetamido-N-(2-aminophenyl)benzamidetrifluoroacetate salt was suspended in a solution of 1:1 EtOH:H2O (320mL). Saturated NaHCO₃ solution (100 mL) was added slowly to adjust thepH to 8. The resultant slurry was stirred for 1.5 hours. Theprecipitates were filtered and washed with water (2×60 mL). After dryingat 40° C. in vacuo for 16 h, 4-acetamido-N-(2-aminophenyl)benzamide(21.3 g, 92% yield, 97.0% HPLC purity) was isolated as crystalline FormB (white solid).

¹HNMR (400 Hz, d⁶-DMSO) δ: 10.22 (s, 1H), 9.58 (s, 1H), 7.94 (d, J=8.8Hz, 2H), 7.70 (d, J=8.8 Hz, 2H), 7.16 (dd, J=1.2 Hz, 8.0 Hz, 1H), 6.97(app dt, J=1.6 Hz, 8.4 Hz, 1H), 6.79 (dd, =1.2 Hz, 8.0 Hz, 1H), 6.6 (appdt, =1.6 Hz, 8.4 Hz, 2H), 4.90 (s, 2H), 2.10 (s, 3H).

Synthesis of Crystalline Tacedinaline Free Base Form A

A sample of 2.0098 g crystalline tacedinaline Forms B and D was placedin 50 mL round bottom flask, and 20 mL of 95:5 EtOH/water added. Thesample was placed in a 70° C. water bath and allowed to warm up. After10 minutes, slurry of solid remains. The sample was seeded with 60.3 mg(3%) Form A. A reflux condenser was attached and the slurry was allowedto stir magnetically at 70° C. for 3 hours. The bath was then cooled to20° C. The cooling process took 1 hour. The sample was then allowed tostir at 20° C. for 1 hour. The slurry was collected by vacuumfiltration, and the solids were placed in a vacuum desiccator at 40° C.for 18 hours. The resulting product was isolated as Form A (white solid,91.8% yield).

¹HNMR (500 Hz, d⁶-DMSO) δ: 10.21 (s, 1H), 9.56 (s, 1H), 7.94 (d, =9 Hz,2H), 7.69 (d, J 6 Hz, 2H), 7.15 (d, J 6, 1H), 6.96 (t, J 9 Hz, 1H), 6.78(d, J=9 Hz, 1H), 6.59 (d, I=6 Hz, 1H), 4.80 (s, 2H), 2.08 (s, 3H)

Example 16 Bioavailability Study of Crystalline Tacedinaline Form A

A study was performed to determine the pharmacokinetics parameters andbioavailability of Tacedinaline Form A in Sprague-Dawley Rats, followinga single intravenous injection (IV) and oral administration (PO).

Formulation:

IV formulation: The test article was dissolved in 10% DMSO/45%PEG400/45% Saline to yield a final concentration of 0.5 mg/ml forintravenous injection. PO formulation: The test article was suspended in0.5% methylcellulose in water to yield a final concentration of 0.2mg/ml for oral administration.

Collection Intervals:

Three rats in each group were used for blood collection at each timepoint: Groups 1 and 2: Pre-dose and post-dose (5 min, 15 min, 30 min, 1hour, 2 hours, 4 hours, 6 hours, 8 hours, and 24 hours).

Analysis Procedure:

The PK blood samples were centrifuged at approximately 8000 rpm for 6minutes at 2-8° C. and the resulting plasma separated and stored frozenat approximately −80° C. (following separation, the plasma may beinitially placed on ice prior to being stored in the −80° C. freezer).All the plasma samples were labeled with detailed information such asstudy number, animal number, matrix, time points of collection and dateof collection.

Pharmacokinetics Analysis:

A standard set of parameters including Area Under the Curve(AUC_((0-t)), and AUC_((0-∞))) elimination half-life (T_(1/2)), maximumplasma concentration (C_(max)), time to reach maximum plasmaconcentration (T_(max)), clearance (CL), and volume of distribution(V_(z)) was calculated using noncompartmental analysis modules in FDAcertified pharmacokinetic program WinNonlin Professional v6.1(Pharsight, USA). Further, the bioavailability was estimated using thefollowing formula:

$F = {\frac{{AUC}_{{({0 - \infty})}{({PO})}} \times {Dose}_{IV}}{{AUC}_{{({0 - \infty})}{({IV})}} \times {Dose}_{({PO})}} \times 100\%}$

The results are summarized below in Tables 16 (IV) and 17 (PO), whichshow selected plasma pharmacokinetics parameters of crystallinetacedinaline Form A in Sprague-Dawley rats following intravenousinjection and oral administration at 1 mg/kg, as well as in FIG. 41which shows the plasma concentration-time curve of the same. This studydemonstrates that crystalline tacedinaline Form A shows 100%bioavailability in rats.

TABLE 16 IV - 1 mg/kg AUC_((0-t)) AUC_((0-∞)) MRT_((0, ∞)) t_(1/2)T_(max) V_(z) CL C_(max) F Animal No. μg/L*hr μg/L*hr hr hr hr L/kgL/hr/kg μ/L % 1 3093.33 3098.83 3.46 2.81 0.50 1.31 0.32 799.48 — 23005.79 3011.53 3.57 2.82 0.50 1.35 0.33 714.90 — Mean 3049.56 3055.183.52 2.81 0.50 1.33 0.33 757.19 —

TABLE 17 PO -1 mg/kg AUC_((0-t)) AUC_((0-∞)) MRT_((0, ∞)) t_(1/2)T_(max) V_(z) _(—) F CL_F C_(max) F Animal No. μg/L*hr μg/L*hr hr hr hrL/kg L/hr/kg μ/L % 1 3697.50 3731.83 4.60 3.81 2.00 1.47 0.27 686.56122.15 2 4016.76 4026.28 4.06 2.90 2.00 1.04 0.25 781.22 131.79 Mean3857.13 3879.06 4.33 3.35 2.00 1.25 0.26 733.89 126.97

-   AUC_((0-t)) Area under the curve from the time of dosing to the last    measurable concentration-   AUC_((0-∞)) Area under the curve from the time of dosing    extrapolated to infinity, based on the last observed concentration-   CL Total body clearance, CL=Dose/AUC-   C_(max) Maximum observed concentration, occurring at T_(max)-   F Bioavailability-   MRT_((0,∞)) Mean residence time from the time of dosing to infinity-   T_(max) Time of maximum observed concentration-   T_(1/2) Terminal half-life=ln(2)λz-   V_(z) Volume of distribution based on the terminal phase

What is claimed is:
 1. A solid form of crystalline4-(acetylamino)-N-(2-aminophenyl)benzamide Form B (tacedinaline Form B),crystalline 4-(acetylamino)-N-(2-aminophenyl)benzamide TFA salt, oramorphous 4-(acetylamino)-N-(2-aminophenyl)benzamide.
 2. Apharmaceutical composition comprising at least one of the solid forms of4-(acetylamino)-N-(2-aminophenyl)benzamide of claim
 1. 3. Apharmaceutical formulation comprising at least one of the solid forms of4-acetylamino)-N-(2-aminophenyl)benzamide of claim 1 and apharmaceutically acceptable carrier.
 4. The pharmaceutical formulationof claim 2 or 3, wherein the solid form is a solid unit dosage form. 5.A pharmaceutical formulation comprising at least one of the solid formsof 4-(acetylamino)-N-(2-aminophenyl)benzamide of claim 1, wherein thesolid form has improved thermal stability relative to tacedinaline FormC.
 6. A pharmaceutical formulation comprising at least one of the solidforms of 4-(acetylamino)-N-(2-aminophenyl)benzamide of claim 1, whereinthe solid form does not contain methanol.
 7. A pharmaceuticalformulation comprising at least one of the solid forms of4-(acetylamino)-N-(2-aminophenyl)benzamide of claim 1, wherein the solidform is more soluble than tacedinaline Form A or tacedinaline Form D. 8.A method of preparing a solid form of4-(acetylamino)-N-(2-aminophenyl)benzamide, comprising preparing any ofcrystalline 4-(acetylamino)-N-(2-aminophenyl)benzamide Form B,crystalline 4-(acetylamino)-N-(2-aminophenyl)benzamide TFA salt, oramorphous 4-(acetylamino)-N-(2-aminophenyl)benzamide, and converting itto a different solid form.
 9. The method of claim 7, wherein crystalline4-(acetylamino)-N-(2-aminophenyl)benzamide TFA salt is prepared andconverted to crystalline 4-(acetylamino)-N-(2-aminophenyl)benzamide FormB.
 10. A method of treating a panic disorder or a post-traumatic stressdisorder in a subject, said method comprising administering crystalline4-(acetylamino)-N-(2-aminophenyl) benzamide Form B to said subject. 11.A method of treating a panic disorder or a post-traumatic stressdisorder in a subject, said method comprising administering apharmaceutical composition comprising crystalline4-(acetylamino)-N-(2-aminophenyl)benzamide Form B to said subject.
 12. Amethod of treating a panic disorder or a post-traumatic stress disorderin a subject, said method comprising administering 0.001 mg/kg to 50mg/kg per day of crystalline 4-(acetylamino)-N-(2-aminophenyl)benzamideForm B to said subject, for at least two consecutive days.
 13. A methodof treating a panic disorder or a post-traumatic stress disorder in asubject, said method comprising administering up to 0.4 mg/kg per day ofcrystalline 4-(acetylamino)-N-(2-aminophenyl)benzamide Form B to saidsubject, for up to 14 consecutive days.
 14. A method of treating a panicdisorder or a post-traumatic stress disorder in a subject, said methodcomprising administering 0.001 mg/kg to 50 mg/kg of crystalline4-(acetylamino)-N-(2-aminophenyl)benzamide Form B to said subject everyother day, every third day, twice a week, or once a week.